Oxygen: a master regulator of pancreatic development?

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1 Biol. Cell (2009) 101, (Printed in Great Britain) doi: /bc Models and Speculations Oxygen: a master regulator of pancreatic development? Scientiae forum Christopher A. Fraker, Camillo Ricordi, Luca Inverardi and Juan Domínguez-Bendala 1 Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136, U.S.A. Biology of the Cell Beyond its role as an electron acceptor in aerobic respiration, oxygen is also a key effector of many developmental events. The oxygen-sensing machinery and the very fabric of cell identity and function have been shown to be deeply intertwined. Here we take a first look at how oxygen might lie at the crossroads of at least two of the major molecular pathways that shape pancreatic development. Based on recent evidence and a thorough review of the literature, we present a theoretical model whereby evolving oxygen tensions might choreograph to a large extent the sequence of molecular events resulting in the development of the organ. In particular, we propose that lower oxygenation prior to the expansion of the vasculature may favour HIF (hypoxia inducible factor)-mediated activation of Notch and repression of Wnt/β-catenin signalling, limiting endocrine cell differentiation. With the development of vasculature and improved oxygen delivery to the developing organ, HIF-mediated support for Notch signalling may decline while the β-catenin-directed Wnt signalling is favoured, which would support endocrine cell differentiation and perhaps exocrine cell proliferation/differentiation. Introduction Our understanding of general development and organogenesis has been characterized by the progressive unravelling of layers of complexity not previously suspected. As occurred with the discovery of the epigenetic code and microrna regulatory networks which changed completely our perspective on the beautifully simple central dogma of molecular biology (Crick, 1958) we are just starting to realize that the molecular changes that define the course of development might also be regulated by physical agents acting upon a background of pre-existing molecular factors. For instance, mechanical forces generated by cell division have been found to be powerful regulators of the balance between proliferation and differentiation (Nelson et al., 2005). Applications of these findings include the manipulation of mesenchymal stem cell fate by the microengineering of tissue shape and tension (Chen, 2004) or the use of cyclic compression to promote chondrogenesis (Huang et al., 2004; Pelaez et al., 2008). Similarly 1 To whom correspondence should be addressed ( Key words: β-cells, oxygen, pancreatic development, Notch, Wnt/β-catenin. Abbreviations used: ARNT, aryl hydrocarbon receptor nuclear translocator; Dvl, Dishevelled; HAT, histone acetyl transferase; HDAC, histone deacetylase; HIF, hypoxia inducible factor; NRX, nucleoredoxin. striking effects on development have been described for other physical parameters, such as bioelectrical fields (Nuccitelli, 1988; Levin, 2003; Robinson and Messerli, 2003) or the nature of the cellular substrate (Czyz and Wobus, 2001; Li et al., 2006; Liu et al., 2006a, 2006b). The exploration of the cellular basis of each one of these phenomena could be the subject of several dedicated reviews. Ours will be focused on oxygen, whose ontogenetic role has already been established in various developmental models. Mutations of several components of the oxygen-sensing machinery, for instance, result in aberrant placental development (Gnarra et al., 1997; Adelman et al., 2000; Cowden Dahl et al., 2005; Takeda et al., 2006). Additional evidence was obtained in vitro by culturing human placental cytotrophoblasts, which proliferate at low oxygen tensions (similar to those found during early pregnancy) but develop an invasive phenotype at higher oxygen concentrations, recapitulating their native behaviour when they populate the maternal spiral arterioles to establish the utero-placental circulation (Genbacev et al., 1997; Zhou et al., 1997; Caniggia et al., 2000). Adipogenesis is also affected by oxygenation, as low oxygen tensions activate HIF (hypoxia inducible factor)-1α, inhibiting differentiation by repressing PPARγ (peroxisome proliferator-activated receptor γ)inadec1 Volume 101 (8) Pages

2 C.A. Fraker and others (deleted in esophageal cancer 1)-dependent fashion (Yun et al., 2002). This effect could not be replicated in HIF-1α-deficient mice. Low oxygen tensions have been associated to improved expansion of stem cells in an undifferentiated state, whereas higher oxygenation tends to induce differentiation (Ezashi et al., 2005; D Ippolito et al., 2006; Zscharnack et al., 2008). Reduced oxygen levels normally stimulate proliferation in adult neural progenitors, but hyperoxia induces maturation in a HIF-1-related manner (Zhu et al., 2005; Burgers et al., 2008; Horie et al., 2008). Beyond a mere proliferation/differentiation switch, a dynamic control of oxygen tension has been shown to provide a fine regulation of cell fate at several levels of neural stem cell development (Chen et al., 2007; Pistollato et al., 2007, 2009). Cardiovascular/ pulmonary development, haematopoiesis and bone morphogenesis are other examples of the critical influence of oxygen in many differentiation processes (Csete, 2005; Simon and Keith, 2008). However, there is an enormous void in our knowledge about the influence of oxygen on pancreatic development, which is otherwise one of the best characterized models of organogenesis. Our interest in the topic arises from our recent finding that oxygen plays a major part on the specification of pancreatic endocrine cells in vitro (Fraker et al., 2007). A superficial examination of the potential mechanisms behind this effect reveals the surprising linkage between the cellular oxygensensing machinery and the molecular networks ultimately responsible for entire developmental programs. Since this is a largely untapped field, our goal is not so much to authoritatively describe a novel regulatory mechanism of pancreatic development as it is to make a first attempt at gathering the pieces of an intriguing biological puzzle. Throughout this review we will try to convey the notion that, when the basic oxygen-sensing biology is confronted with key milestones of pancreatic development, a clear picture starts to emerge. Pancreatic development Despite some timing differences (Piper et al., 2004), there is a high degree of conservation between human and mouse pancreatic development. The latter is initiated around E8.5 from a region of the foregut epithelium where Shh (sonic hedgehog) signalling has been down-regulated. The pancreatic primordia (dorsal and ventral) are characterized by the transient expression of the genes Pdx1 and Ptf1α (Kumar and Melton, 2003). Epithelial cells at this stage might be considered equipotent, and their proliferation results from a molecular equilibrium barely maintained by active Notch signalling (Kadesch, 2004). The rupture of this unstable condition results in the first fate decisions: cells subjected to persistent Notch signalling will remain undifferentiated and cycling; down-regulation of Notch, in contrast, will yield cells that express the pro-endocrine gene Ngn3 (Apelqvist et al., 1999). In accordance to the changing signalling microenvironment, these Ngn3-positive cells will differentiate into all the endocrine cell types throughout development: α (glucagon-producing)cells from E9.5; β (insulin-producing) cells from E ; δ (somatostatin-producing) cells from E13.5 and PP (pancreatic polypeptide) cells shortly before birth. As for the exocrine lineage, CRE-ER TM tracing experiments show that acinar cells arise from Pdx1+ progenitors in which neither Notch signalling nor Ptf1α expression are down-regulated (Gu et al., 2002, 2003). The proliferation of exocrine progenitor cells observed at a later point has been associated with sustained Wnt/β-catenin signalling (Murtaugh et al., 2005; Wells et al., 2007). Therefore, it can be said that the proliferation/differentiation balance of the two major pancreatic lineages is regulated to a significant extent by either Notch (endocrine/early exocrine progenitor cell proliferation) or Wnt/β-catenin (late exocrine progenitor cell proliferation). The secondary transition starts around E13.5 and is maintained throughout intrauterine development (Pictet and Rutter, 1972; Pictet et al., 1972). This phase is characterized by a massive wave of endocrine cell differentiation, with a strong reactivation of Pdx1 in newly formed β-cells. An interesting observation that we will revisit later is that this phenomenon is coincident in time with the initiation of blood flow (and therefore higher oxygenation) within the organ (Colen et al., 1999). Effects of oxygen on endocrine/exocrine specification in vitro Despite the major influence of oxygen on pancreatic islet survival and function (Chase et al., 1979; Berggren, 1981; Papas et al., 1996; Kazzaz et al., 1999; Carlsson and Mattsson, 2002; Carlsson et al., 2002, 2003; Ko et al., 2008), the first systematic studies on its participation in the development 432 C The Authors Journal compilation C 2009 Portland Press Ltd

3 Oxygen and pancreatic development Scientiae forum of the pancreas were reported only recently (Fraker et al., 2007). The apparent oversight might have been due to the inability of standard culture mechanisms to deliver oxygen in a physiological fashion, which has complicated the appropriate design of in vitro studies. This limitation was finally overcome with the development of novel culture vessels designed to minimize the formation of gas diffusion gradients, maintaining relatively constant oxygen levels throughout cellular aggregates (Fraker et al., 2007). These culture devices are generally based on the replacement of the plastic bottom of a flask or dish by air-permeable membranes. By culturing the cells atop these vessels, they receive oxygen both from the top (via diffusion through the culture medium) and the bottom (through the breathable membrane). Variations of this concept were previously used to increase pancreatic islet culture density without loss of viability and function (Papas et al., 2005) and to maintain long-term hepatocyte phenotype in culture (De Bartolo et al., 2006). Using an evolved system based on the use of perfluorocarbon in the basal membrane, Fraker et al. (2007) exposed E13.5 mouse pancreatic buds (at the initiation of the secondary transition) to physiologically high oxygenation (Carlsson and Mattsson, 2002) for three days. The differentiation outcome was compared with that of control explants, grown in regular (hypoxic) conditions. Two significant effects were observed in the experimental group: (a) faster proliferation of non-endocrine progenitor cells; and (b) enhanced endocrine differentiation. The profile of pancreatic buds cultured in this way was largely indistinguishable from that observed during normal native development at corresponding time points, suggesting that physiological oxygenation is critical for appropriate pancreatic development. Although there is no literature yet on the effects of oxygenation on pancreatic development in vivo,these results are consistent with those of preliminary experiments in which exposure of pregnant mice to hyperbaric oxygen led to an acceleration of pancreatic, but not overall embryo, development (C.A. Fraker, S. Álvarez, L. Inverardi, C. Ricordi and J. Dominguez- Bendala, unpublished work). The oxygen-sensing machinery and pancreatic development Cells respond to low oxygen concentrations by activating the HIF-1 pathway. HIF-1 is a heterodimer consisting of two basic helix-loop-helix/pas protein subunits: (a) the aryl hydrocarbon nuclear translocator (ARNT or HIF-1β), which is constitutively expressed in an oxygen-independent manner; and (b) the oxygen-dependent α domain (Pugh and Ratcliffe, 2003). Evidence also indicates that human islet expression of ARNT/HIF-1β expression is dramatically reduced in Type 2 diabetes, and β-cellspecific mutations in HIF-1β lead to impaired glucose tolerance and insulin secretion (Gunton et al., 2005; Levisetti and Polonsky, 2005; Czech, 2006). As for the α domain, HIF-1α is the most widely expressed member of its family in mammals, and knockouts exhibit embryonic lethality (Iyer et al., 1998). HIF-2α and HIF-3α are more specialized and have more tissue-specific functions (Semenza, 2000; Maynard et al., 2003, 2007; Wiesener et al., 2003; Warnecke et al., 2004; Chavez et al., 2006). Although HIF-2α is also highly expressed in the pancreas (Wiesener et al., 2003), there are no reports suggesting that its function in this organ is dissimilar to that of HIF-1α. Therefore, for the sake of simplicity, we will base our models on the latter. In normoxia, HIF-1α is polyubiquitinatedand targeted for degradation by an E3 ubiquitin ligase complex that contains, among other protein associates, the tumour suppressor protein pvhl (von Hippel Lindau protein). Hypoxia, however, stabilizes the protein (Pugh and Ratcliffe, 2003; Bell et al., 2005), allowing it to interact with downstream genes involved in processes such as angiogenesis (Pugh and Ratcliffe, 2003; Diez et al., 2007), erythropoiesis (Stockmann and Fandrey, 2006), glucose transport/glycolysis (Wenger, 2002), apoptosis (Moritz et al., 2002) and stem cell self-renewal/proliferation (Jogi et al., 2002; Ezashi et al., 2005; Gustafsson et al., 2005; Diez et al., 2007). The Notch pathway is generally involved in proliferation and the maintenance of an undifferentiated state (Kadesch, 2004), and its down-regulation is the first step in the initiation of the pancreatic endocrine differentiation cascade (Apelqvist et al., 1999; Jensen et al., 2000; Lammert et al., 2000; Edlund, 2001; Hart et al., 2003; Murtaugh et al., 2003). HIF-1α is known to interact with the intracellular domain of Notch, activating this pathway under hypoxic conditions (Cejudo-Martin and Johnson, 2005; Gustafsson et al., 2005; Pearand Simon, 2005; Sainson and Harris, 2006; Diez et al., 2007). This is in agreement Volume 101 (8) Pages

4 C.A. Fraker and others with the observation that hypoxic areas during embryonic development are generally associated with proliferation (Lee et al., 2001). In this context, one potential explanation of the in vitro effects of oxygen on endocrine differentiation is that high oxygen tensions de-activate HIF-1α, which in turns results in an overall down-regulation of Notch signalling and an outburst of endocrine cell differentiation. But what is high oxygen during pancreatic development in vivo? It is generally accepted that mammalian development occurs at very low oxygen levels prior to the onset of blood circulation (Mitchell and Yochim, 1968; Rodesch et al., 1992; Simon and Keith, 2008). In the rat embryo, the yolk sac becomes the first important organ of O 2 /CO 2 exchange around E10.5 (New, 1978). Prior to that, the embryo receives oxygen mainly through diffusion (New, 1978; Miki et al., 1988). Even in full environmental oxygenation ( po 2 of 160 mmhg), the maximal tissue volume that can be oxygenated by simple diffusion is approx. 1mm 3 (Burggren, 2004). If we factor in the observation that the po 2 of the mammalian reproductive tract is less than half that of the atmosphere, the upper limit of diffusion in the pre-circulation embryo is even smaller (Gassmann et al., 1996). Therefore, it is reasonable to expect that pancreatic development occurs at oxygen concentrations barely above anoxia, at least until the advent of blood flow in the organ at E13.5 (Colen et al., 1999). In the absence of direct measurements of pancreatic oxygen tension in the embryo, we can speculate that the above event will mark a turning point in the availability of oxygen to the tissue. Thus, physiological oxygen concentrations are expected to be low prior to E13.5 and high afterwards, which is in line with the developmental patterns observed before (Notch-dependent expansion of endocrine progenitors) and during (endocrine cell differentiation) the secondary transition. Since blood vessel formation is promoted by HIF-1 under hypoxic conditions (Pugh and Ratcliffe, 2003; Diez et al., 2007), vasculogenesis/blood flow could be considered both cause and consequence of pancreatic development. Of course, developmental events rarely surprise us with single explanations. Higher oxygenation may also prevent the differentiation and promote the proliferation of certain cell types, which would be difficult to explain by the HIF-1/Notch interaction. Of particular interest for pancreatic development is the potential role of oxygen in the Wnt pathway. While Wnt signalling has been traditionally divided in canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) from a ligand point of view (i.e. some Wnt ligands will lead to β-catenin accumulation, whereas others act through other mechanisms), a more recent reassessment of the evidence suggests that Wnt ligands are not intrinsically canonical or non-canonical, but that they operate through different pathways depending on the nature of their receptors (van Amerongen et al., 2008). In the Wnt/β-catenin pathway, when ligands bind to the Frizzled receptor, the β-catenin destruction complex is inhibited. Accumulated β-catenin migrates subsequently to the nucleus, where it binds to TCF (T-cell factor)/lef (lymphoid enhancer factor) complexes to activate target genes normally involved in a variety of processes, including proliferation (Hoppler andkavanagh, 2007). Wnt/β-catenin activation has been shown to be essential for the maintenance of undifferentiated proliferating exocrine progenitor cells in the developing pancreas (Murtaugh et al., 2005; Wells et al., 2007), and there is significant evidence that Wnt/β-catenin signalling is inhibited in hypoxia. For instance, HIF-1α has been shown to compete with TCF4 for direct binding to β-catenin (Kaidi et al., 2007). Also, the redox-regulating protein NRX (nucleoredoxin) inhibits Wnt/β-catenin signalling by binding to Dvl (Dishevelled), a critical component of the pathway (Funato et al., 2006). However, the oxidation of NRX results in its dissociation from Dvl, which results in a net downregulation of Wnt/β-catenin activity (Figure 1). Therefore, in specific cell types (such as exocrine progenitor cells), high oxygen may promote proliferation through Wnt/β-catenin activation. Summarizing what has been proposed so far, the hypoxia-induced initiation of blood flow in the pancreas would: (a) down-regulate Notch signalling in endocrine progenitors, promoting their differentiation into islet endocrine cell types; (b) up-regulate Wnt/β-catenin signalling in exocrine progenitor cells, taking over Notch to stimulate their proliferation throughout the rest of development. It is important to stress that, even though both the endocrine and the exocrine compartments are differentiating and expanding during the same developmental windows (which would argue against the differential impact of higher oxygenation), the mechanisms by which 434 C The Authors Journal compilation C 2009 Portland Press Ltd

5 Oxygen and pancreatic development Scientiae forum Figure 1 Notch and Wnt/β-catenin pathways are differentially affected by oxygen levels In low oxygen concentrations (left), HIF-1α is stable and dimerizes with HIF-1β. The complex is known to bind to the intracellular domain of Notch (bottom; interaction represented by an arrow), favouring the transcription of target genes. However, higher oxygen levels (right) induce the degradation of HIF-1α by the E3 ubiquitin ligase complex, decreasing the net activity of Notch. Upon activation of the Wnt receptor (top), β-catenin destruction is halted and the protein migrates to the nucleus, where it forms a complex with other proteins to activate target genes. Low oxygen conditions (left) favour the sequestration of Dvl (an essential component of the pathway) by NRX. In addition, stabilized HIF-1α will compete with β-catenin co-factors. Hypoxia, therefore, is associated with an overall down-regulation of the pathway. In contrast, higher oxygen tensions (right) will (a) oxidize NRX, releasing Dvl; and (b) destabilize the HIF-1 complex, eliminating the competition for β-catenin co-factors. A high oxygen concentration is thus conducive to enhanced Wnt/β-catenin activity. they do so are not the same and may have different kinetics. As mentioned earlier, an up-regulation of any given signalling mechanism will have different effects depending on the receptors expressed in the different cell subsets (van Amerongen et al., 2008). Endocrine cells arise from pancreatic progenitors in a Notch-dependent fashion at early stages of development (Apelqvist et al., 1999), when oxygen tensions are low. Upon vascularization and in the presence of high oxygenation, they progressively start to increase their numbers by proliferation of differentiated cells (Zhang et al., 2006). This would be consistent with the recent observation that Wnt signalling (which is up-regulated in high oxygen) has been associated with increased β-cell proliferation (Liu and Habener, 2008). Exocrine cells behave in a similar way prior to the secondary transition, but their main expansion, which is believed to occur through progenitor cells and not by self-replication, occurs in a Wnt-mediated manner from the onset of the secondary transition onwards (Murtaugh et al., 2005; Wells et al., 2007). Thus, the increase in Wnt activity associated with higher oxygen availability might have the twofold effect of increasing (a) endocrine cell mass by selfreplication and (b) exocrine cell mass by progenitor cell expansion. Oxygen may have an additional function during pancreatic development, one even subtler than that Volume 101 (8) Pages

6 C.A. Fraker and others of modifying the general activity of large signalling pathways such as Notch and Wnt/β-catenin. We are referring to its direct influence on local chromatin reorganization, which may lead to the expression or silencing of genes critically involved in the progression of pancreatic development. Among the many post-translational modifications of the nucleosome that affect DNA compactness, and therefore accessibility of the transcriptional machinery, one of the best characterized is lysine acetylation. The net level of acetylation at any given locus is the result of the balance between HATs (histone acetyl transferases) and HDACs (histone deacetylases). Highly acetylated loci (with high HAT activity) have a looser DNA coiling, which is typically indicative of elevated transcription. Strong HDAC activity, in contrast, is likely to lead to gene silencing. HDACs 1, 2, 3, 4 and 7 are known to interact with HIF-1α, and hypoxic conditions have been unequivocally linked to enhanced nuclear HDAC-mediated transcriptional repression (Simon and Keith, 2008). Indeed, the promoter of NeuroD/Beta2, a downstream target of Ngn3 and fundamental component of the pancreatic β-cell differentiation cascade (Naya et al., 1997), is transcriptionally repressed by a protein complex that includes HDACs 1 and 3 (Liu et al., 2006c). Intrauterine growth retardation, which is commonly associated with lower oxygen transport to the uteroplacental unit (Martin-Gronert and Ozanne, 2007), results in the progressive epigenetic silencing of Pdx1 through the recruitment of HDAC-1 to its promoter (Park et al., 2008). With both NeuroD/Beta2 and Pdx1, transcriptional repression could be reversed by HDAC inhibition (Liu et al., 2006c; Park et al., 2008). A recent study on explanted murine embryonic pancreatic rudiments found that treatment with different HDAC inhibitors enhanced ductal at the expense of acinar differentiation, upregulated the Ngn3-positive pro-endocrine lineage and enhanced the pool of β-cells (Haumaitre et al., 2008). Additional indirect evidence on the importance of HDAC down-regulation for the progression of pancreatic development comes from in vitro differentiation experiments where HDAC inhibitors were successfully used to induce the expression of essential markers of pancreatic development (Goicoa et al., 2006; Jiang et al., 2007). The study of the many other cellular processes affected by oxygen-regulated chromatin reorganization, including DNA repair (Mihaylova et al., 2003), inflammation (Rahman et al., 2004; Rahman and Adcock, 2006), senescence (Dohi et al., 2008) or other types of differentiation (Yun et al., 2005), is obviously beyond the scope of this review. In the case of pancreatic development, the evidence gathered so far suggests that high oxygen conditions present after E13.5 may favour reorganization by depleting HDACs from protein complexes that repress the expression of relevant genes. Another indirect effect of oxygen in shaping overall pancreatic development is observed through the HIF-1α-dependent formation of vasculature. In vitro experiments showed that blood vessel endothelium is capable of inducing expression of the insulin gene in explanted mouse embryonic endoderm. Vascularization in the posterior foregut in transgenic mice expressing VEGF (vascular endothelial growth factor) under the control of the Pdx1 promoter led to ectopic insulin expression and islet hyperplasia. Conversely, removal of the developing dorsal aorta in frog embryos abrogated insulin expression in vivo (Lammert et al., 2001). Subsequent studies demonstrated that endothelial cell signals from the aorta were instrumental in the earlier stages of pancreatic development, up-regulating the expression of the critical pancreatic morphogen p48/ptf1a (Lammert et al., 2003; Yoshitomi and Zaret, 2004). The theoretical interaction of all the above pathways is depicted in Figure 2. This model conforms to the evidence presented herein, but we propose it only as a working hypothesis that needs to be experimentally tested. In this context, the recent availability of floxed HIF-1α mice (Ryan etal., 2000) mighthelpus explore in detail the effect of variable oxygen tensions on individual cellular subsets within the developing pancreas. A view of oxygen as the top ruler of all these molecular networks would be overly simplistic. In fact, we must emphasize that, whereas mechanical and other external factors do play an important role in development, their input is based on the availability of molecular factors capable of replying to such input within a window of developmental potential. However, a deeper knowledge of the influence that physical variables have on fundamental cell processes might lead to the realization that there is yet another level of regulation orchestrating gene expression, cell behaviour and, ultimately, the unfolding of 436 C The Authors Journal compilation C 2009 Portland Press Ltd

7 Oxygen and pancreatic development Scientiae forum Figure 2 A theoretical framework to explain the role of oxygen in pancreatic development Hypoxic conditions present in the pancreatic buds prior to the initiation of blood flow (E8.5 E13.5, top) will lead to the stabilization of HIF-1α, which in turn will (a) activate Notch, repressing endocrine differentiation from multipotential progenitors; (b) induce the expression of VEGF; and (c) bind to HDACs to repress target genes such as Pdx1, Ngn3 and Beta2/NeuroD. The Wnt/β-catenin pathway remains down-regulated under hypoxic conditions. This developmental setting (high Notch and low Wnt/βcatenin activity) results in multipotential progenitor cell proliferation and repression of endocrine differentiation. VEGF synthesis as a result of HIF-1 activity is a main determinant of vascularization. Blood starts to flow in the pancreatic buds around E13.5, with a consequent increase in the availability of oxygen (bottom). Under these conditions: (a) HIF-1α will be degraded, slowing down Notch activity and preventing the HDAC-mediated repression of target genes; and (b) the blockages on the Wnt/β-catenin pathway are removed. This setting (low Notch and high Wnt/β-catenin activity) favours endocrine differentiation and exocrine precursor cell proliferation. embryonic development. This putative physiological code might not just overlap known regulatory pathways, but perhaps even override them under certain circumstances. Deciphering such a code would be of enormous interest not only from a basic science perspective but also for biomedical purposes -where the discovery of another level of complexity might paradoxically result in an overall simplification of the problem. Funding Our work is or has been funded by the American Diabetes Association (ADA) [grant number 1-04-ISLET- 18], the William H. Wallace Coulter Foundation and the Diabetes Research Institute Foundation (DRIF). References Adelman, D.M., Gertsenstein, M., Nagy, A., Simon, M.C. and Maltepe, E. (2000) Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses. Genes Dev. 14, Apelqvist, A., Li, H., Sommer, L., Beatus, P., Anderson, D.J., Honjo, T., Hrabe de Angelis, M., Lendahl, U. and Edlund, H. (1999) Notch signalling controls pancreatic cell differentiation. Nature 400, Bell, E.L., Emerling, B.M. and Chandel, N.S. (2005) Mitochondrial regulation of oxygen sensing. Mitochondrion 5, Berggren, P.O. (1981) Characteristics of Ba 2+ -stimulated insulin release with special reference to pancreatic β-cells sensitized by cyclic AMP. Acta Biol. Med. Ger. 40, Volume 101 (8) Pages

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